International Journal of Molecular Sciences

Review Molecular Markers of Therapy-Resistant Glioblastoma and Potential Strategy to Combat Resistance

Ha S. Nguyen 1,2, Saman Shabani 1, Ahmed J. Awad 1,3, Mayank Kaushal 1 ID and Ninh Doan 1,4,* 1 Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, WI 53226, USA; [email protected] (H.S.N.); [email protected] (S.S.); [email protected] (A.J.A.); [email protected] (M.K.) 2 Faculty of Neurosurgery, California Institute of Neuroscience, Thousand Oaks, CA 91360, USA 3 Faculty of Medicine and Health Sciences, An-Najah National University, Nablus 11941, Palestine 4 Department of Neurosurgery, Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36688, USA * Correspondence: [email protected]



Received: 24 May 2018; Accepted: 13 June 2018; Published: 14 June 2018


Abstract: Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system. With its overall dismal prognosis (the median survival is 14 months), GBMs demonstrate a resounding resilience against all current treatment modalities. The absence of a major progress in the treatment of GBM maybe a result of our poor understanding of both GBM tumor biology and the mechanisms underlying the acquirement of treatment resistance in recurrent GBMs. A comprehensive understanding of these markers is mandatory for the development of treatments against therapy-resistant GBMs. This review also provides an overview of a novel marker called acid ceramidase and its implication in the development of radioresistant GBMs. Multiple signaling pathways were found altered in radioresistant GBMs. Given these global alterations of multiple signaling pathways found in radioresistant GBMs, an effective treatment for radioresistant GBMs may require a cocktail containing multiple agents targeting multiple cancer-inducing pathways in order to have a chance to make a substantial impact on improving the overall GBM survival.

Keywords: glioblastoma; acid ceramidase; acid ceramidase inhibitors; carmofur; radioresistance; radiation; sphingosine; sphingosine-1-phosphate; S1P

1. Introduction Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system. With its overall dismal prognosis, GBMs demonstrate a resounding resilience against all current treatment modalities. The estimated overall survival of GBM patients is less than 1.5 years, and the 5-year survival rate is 5% [1–3]. The median age of diagnosis of GBM has increased to 64 years over the last decades, and the top incidence is 15.24/100,000 populations diagnosed within the age range of 75–84 years [1–3]. While radiation is the only proved cause of GBM, only a minority of patients develop GBMs following exposure to radiation [4]. The etiology of GBM remains to be discovered, and fewer than 5% of patients have a germline mutation which increases the risk for developing GBMs [5,6]. Symptoms at presentation are based on the location of GBMs. Eloquent-area tumors often engender symptoms ranging from numbness, weakness, and visual disturbance to language deficits, while tumors in other areas (including the non-dominant frontal and temporal lobes or the corpus callosum) may induce non-specific symptoms (such as seizures, which can be controlled with anticonvulsant medications in 25% of patients with newly diagnosed GBMs [7]). However, new

Int. J. Mol. Sci. 2018, 19, 1765; doi:10.3390/ijms19061765 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 1765 2 of 23 emerging data have suggested that the administration of anticonvulsants may not be beneficial and can produce significant, undesired effects in GBM patients without seizures [8,9]. The presenting symptoms include headaches (~60%), memory loss (~40%), and cognitive, language, or motor deficits (~40%) [10]. The most common imaging modality to diagnose GBMs is magnetic resonance imaging (MRI) of the brain with and without gadolinium contrast. A heterogeneous ring-enhancement with area of central necrosis is the signature feature of GBMs; infrequently, GBMs can be multi-focal. Headache has been attributed to peritumoral edema, which can cause a major midline shift or mass effect [11]. Steroids such as dexamethasone are commonly employed to provide relief from headache or deficits by reducing the peritumoral edema, generally within 48 h [12,13]. Another therapy aimed at reducing the peritumoral edema is based on the anti-angiogenesis antibody bevacizumab, but it has been shown not to affect the overall survival in patients with newly diagnosed GBMs [14,15]. GBMs having certain prognostic biomarker mutations, such as isocitrate dehydrogenase (IDH), may present, on MRI, with characteristic features, such as a large non-enhancing mass with pial invasion, decreased blood flow, minimal edema and necrosis, and a tendency for the frontal and temporal lobes [16,17]. Following surgery, resected GBM tissues are formalin-fixed and paraffin-embedded prior to undergoing histopathology examinations, which characteristically show palisading necrosis, marked pleomorphism, a high mitotic index, and microvascular proliferation. Additionally, these GBM tissues are also further examined by immunostaining or sequencing for IDH mutations, O6-methylguanine methyltransferase (MGMT) methylation, and other prognostic biomarkers, which will be discussed in detail below [18,19]. The absence of a major progress in the treatment of GBM may be a result of our poor understanding of both GBM tumor biology and the mechanisms underlying the acquirement of treatment resistance in recurrent GBMs. Others have proposed that glioblastoma stem-like cells (GSCs), carrying the cell membrane marker CD133, may play a significant role in the resistance of this cancer to chemotherapy and radiotherapy [20–23]. The higher expression levels of CD133 have been linked to poorer prognosis [23]. Proteins or signaling pathways that maintain stemness may contribute to the development of therapy-resistant GBMs [24]. Novel druggable targets that have been reported to combat therapy-resistant GBMs include sodium pump α1 subunit, wingless-type MMTV integration site family member (Wnt)/β-catenin, sonic hedgehog/Glioma-associated oncogene (SHH/GLI), oligodendrocyte transcription factor 2(OLIG2), polycomb group RING finger protein 4 (BMI1), NANOG, and inhibitor of differentiation/DNA binding (ID1)[24,25]. More recently, circular RNAs (circRNAs) such as circSMARCA5, whose expression is downregulated in GBM samples as compared to control tissues, has been described to function as a novel tumor-suppressor, regulating the migration of GBM cells by modulating the oncoprotein that modulates cell migration, called RNA binding protein serine- and arginine-rich splicing factor 1 (SRSF1) [26]. A comprehensive understanding of established prognostic markers is mandatory for the development of treatments against therapy-resistant GBMs. In addition to discuss the established prognostic markers, this review also provides an overview of a novel marker called acid ceramidase (ASAH1) and its implications in the development of radioresistant GBMs. Multiple signaling pathways were found altered in radioresistant GBMs. Given the global alterations of multiple signaling pathways found in radioresistant GBMs, an effective treatment targeting radioresistant GBMs may require a cocktail containing multiple agents targeting multiple cancer-inducing pathways in order to have a chance to make a substantial impact on improving overall GBM survival.

2. O6-Methylguanine Methyltransferase (MGMT) Alkylating agents, such as temozolomide (TMZ), attach an alkyl group to the DNA, frequently at the N-7 or O-6 positions of guanine residues (Figure1)[ 27]. This process damages the DNA and triggers cell cycle death, unless the DNA is promptly repaired. O6-methylguanine methyltransferase (MGMT), a DNA repair protein, can hydrolyze the alkyl groups off guanine and impede the effectiveness of such chemotherapeutic agents [28]. Methylation of the MGMT promoter at CpG sites can suppress Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 21 effectivenessInt. J. Mol. Sci. of2018 such, 19, chemotherapeut 1765 ic agents [28]. Methylation of the MGMT promoter at CpG sites3 of 23 can suppress the gene and promote sensitivity to alkylating agents. Overall, up to 45–47% of GBMs exhibited methylation in prior studies [28,29]. The MGMT methylation status of tumors significantly correlatesthe gene with and progression-free promote sensitivity survival to alkylating (PFS) an agents.d overall Overall, survival up (OS) to 45–47% for patients of GBMs undergoing exhibited treatmentmethylation with in alkylating prior studies agents [28 ,[28].29]. TheFor MGMTnewly diagnosed methylation GBM, status alkylating of tumors agents significantly are a mainstay correlates optionwith progression-freeirrespective of the survival MGMT (PFS) methylation and overall stat survivalus [30]; (OS) for forelderly patients patients, undergoing those with treatment MGMT with methylationalkylating agentsmay have [28]. a Forgreater newly benefit diagnosed from TMZ GBM, monotherapy alkylating agents than are radiotherapy a mainstay option[31], while irrespective those withoutof the MGMTmethylation methylation may not status benefit [30 ];from for elderlyalklylat patients,ing agents. those At withrecurrence, MGMT alterations methylation in mayMGMT have methylationa greater benefit status fromhave TMZnot been monotherapy detected [32]; than moreover, radiotherapy the relationship [31], while with those MGMT without methylation methylation persistsmay not at benefit recurrence, from alklylating and TMZ agents. re-challenge At recurrence, is alterationsa sensible in MGMTchoice methylationfor patients status with have MGMT-methylatednot been detected [GBM32]; moreover, [33]. the relationship with MGMT methylation persists at recurrence, and TMZ re-challenge is a sensible choice for patients with MGMT-methylated GBM [33].

Figure 1. A cartoon showing temozolomide (TMZ) alkylating DNA at the O-6 position of guanine Figure 1. A cartoon showing temozolomide (TMZ) alkylating DNA at the O-6 position of guanine residues and the removal of this alkyl group by O6-methylguanine methyltransferase (MGMT) through residues and the removal of this alkyl group by O6-methylguanine methyltransferase (MGMT) a DNA repair process. through a DNA repair process.

Presently,Presently, molecular molecular testing testing occurs occurs via via either either quantitative quantitative methylation-specific methylation-specific PCR PCR or or pyrosequencingpyrosequencing [34]. [34]. The The former former employs employs methylation-specificmethylation-specific primer primer pairs pairs to to probe probe CpG CpG islands islands with withhigh high methylation methylation density; density; the latterthe latter enumerates enumerat thees methylation the methylation sites atsites individual at individual CpG sites CpG through sites throughthe “sequencing-by-synthesis” the “sequencing-by-synthesis” principle principl when nucleotidese when nucleotides get incorporated get incorporated by DNA polymerase by DNA [35 ]. polymeraseTo complicate [35]. matters, To complicate there is matters, no accepted there threshold is no accepted for the threshold number of for methylated the number sites of formethylated a tumor to sitesbe classifiedfor a tumor as to “methylated”; be classified variousas “methylated” detection; methodsvarious detection yield methylation methods ratesyieldvarying methylation from rates 33% to varying60% for from the 33% same to group 60% for of GBMthe same patients group [36 of]. GBM Moreover, patients additional [36]. Moreover, research additional is required research to elucidate is requiredthe impact to elucidate of the extent the impact of methylation of the extent or the of patterns methylation of methylation or the patterns on GBM of methylation survival [35 on]. GBM survival [35]. 3. Epidermal Growth Factor Receptor (EGFR) 3. EpidermalEpidermal Growth growth Factor factor Receptor receptor (EGFR) (EGFR) is a transmembrane tyrosine kinase that functions as a critical player in pathways linked to cell proliferation, migration, and survival. EGFR activity can be Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase that functions as augmented via gene amplification or EGFR variant III deletion mutation (EGFRvIII); the latter results a critical player in pathways linked to cell proliferation, migration, and survival. EGFR activity can in a truncated receptor that is constitutively active, promoting mitogenic cascades. EGFR amplification be augmented via gene amplification or EGFR variant III deletion mutation (EGFRvIII); the latter occurs in roughly 40–60% of GBM; EGFRvIII, which only occurs in a subset of those GBMs with results in a truncated receptor that is constitutively active, promoting mitogenic cascades. EGFR EGFR amplification, arises in approximately 20–30% of GBM overall [35,37–40]. EGFR amplification amplification occurs in roughly 40–60% of GBM; EGFRvIII, which only occurs in a subset of those is assessed via fluorescence in situ hybridization (FISH); EGFRvIII expression can be established by GBMs with EGFR amplification, arises in approximately 20–30% of GBM overall [35,37–40]. EGFR immunohistochemistry (IHC) [38]. amplification is assessed via fluorescence in situ hybridization (FISH); EGFRvIII expression can be Studies regarding the implications of EGFR amplification and EGFRvIII mutation have reported established by immunohistochemistry (IHC) [38]. mixed, conflicting results regarding GBM survival [27,37,39]. Given the positive results in other types of Studies regarding the implications of EGFR amplification and EGFRvIII mutation have cancers, researchers believed that receptor tyrosine kinase inhibitors could play a role in the treatment reported mixed, conflicting results regarding GBM survival [27,37,39]. Given the positive results in of GBM [39]. However, clinical trials for GBM designed to target EGFR have been disappointing [39]. other types of cancers, researchers believed that receptor tyrosine kinase inhibitors could play a role The dearth of clinical effectiveness may be due to the inability of the examined drugs to cross the in the treatment of GBM [39]. However, clinical trials for GBM designed to target EGFR have been blood-brain barrier and/or the development of resistance through gained mutations [40]. disappointing [39]. The dearth of clinical effectiveness may be due to the inability of the examined drugs4. Isocitrate to cross Dehydrogenase the blood-brain (IDH barrier)1/2 and/or the development of resistance through gained mutations [40]. Isocitrate dehydrogenase (IDH) is a component of the Krebs cycle that converts isocitrate and cofactor NAD+ to carbon dioxide, NADH, and α-ketoglutarate. Since the initial discovery of IDH

Int. J. Mol. Sci. 2018, 19, 1765 4 of 23 mutations in GBM [41], several studies have observed that IDH mutations occurred in approximately 8–13% of all GBMs, including greater than 80% of secondary GBM [42]. The most common mutations are IDH1 R132 and IDH2 R172, comprising roughly 90% of IDH mutations; the former is noted in more than 70% of grade 2/3 gliomas and in GBMs that progressed from these lower-grade tumors [41,43]. The mutations cause a buildup of the onco-metabolite D-2-hydroxy-glutarate, which can disturb DNA methylation, gene transcription, and histone alterations; moreover, mutations may decrease NAPDH formation, promoting oxidative stress and leading to DNA damage [27,35]. Several studies have documented survival benefits (OS and PFS) in gliomas with IDH mutations, ranging from an average of 12 to 30 months [27,42–44]. In addition, IDH mutations convey a higher sensitivity to TMZ and radiotherapy [45–47]. IDH mutations have also been correlated with improved MRI-defined enhancing disease, allowing larger resections [48]. At present, IDH mutations can be detected via sequencing or IHC [38]. Preclinical studies have demonstrated that small molecule inhibitors of mutant IDH can lower the intracellular levels of D-2-hydroxy-glutarate, overturn epigenetic dysregulation, and promote cellular differentiation [49].

5. 1p19q Co-Deletion The unbalanced whole-arm translocation of the centromeric portions between chromosomes 1q and 19q is defined as 1p19q co-deletion. The recent WHO 2016 criteria utilize this co-deletion, along with an IDH mutation, to classify gliomas into the oligodendroglial phenotype. The co-deletion occurs in roughly 60–80% of grade 2 or 3 oligodendrogliomas, 20–50% of grade 2 or 3 oligoastrocytomas, and less than 10% of diffuse gliomas (together with GBM) [50]. For oligodendroglioma, this co-deletion has been associated with favorable survival as well as responsiveness to chemotherapy (PCV and temozolomide) and radiotherapy [51–54]. The reasoning behind this sensitivity to treatment remains elusive. Studies concerning the co-deletion in GB have reported mixed results [55,56]; however, a meta-analysis by Zhao et al. [57], incorporating 3408 gliomas across 28 studies, noted that 1p/19q co-deletion was associated with improved survival (PFS and OS) irrespective of the histological grade. Frequently, detection of the 1p19q co-deletion is completed through FISH; other methods include microsatellite analysis, PCR, and array comparative genomic hybridization [58].

6. α-Thalassemia/Mental Retardation Syndrome X-Linked (ATRX) The ATRX (α-thalassemia/mental retardation syndrome X-linked) gene encodes a protein involved in genomic stability, chromatin remodeling, and DNA methylation [59]. Inactivation of the gene is highly linked to the ALT (alternative lengthening of telomeres) phenotype. ALT is a mechanism for the regulation of telomere length that is vital to cell survival and proliferation [59]. Its role in glioma biology has only recently been explored. ATRX mutation is frequently associated with IDH mutations, but rarely with 1p19q co-deletions [59]. For anaplastic gliomas, ATRX loss defines a subset of IDH mutants with a significantly longer median time to treatment failure (close to 24 months) [60]. By using a mouse model of ATRX-deficient GBM, Koschmann et al. suggested that ATRX mutations lead to a genetically erratic tumor. With no treatment, the tumor behaved rather aggressively; on the contrary, with treatment directed at double-stranded DNA damage, the overall survival improved [61]. Commonly, detection of ATRX loss is performed via IHC; other methods include PCR, sequencing, and Western blotting [35,38].

7. Telomerase Reverse Transcriptase (TERT) Telomeres are nucleoprotein complexes (comprised of hundreds of repetitive nucleotide sequences) that bind the extremes of chromosomes to ensure chromosomal integrity [62]. Each cell division prompts telomere truncation until its depletion, which provokes cell dormancy or death [62]. Telomerase reverse transcriptase (TERT) is a subunit of telomerase, an enzyme that inserts additional nucleotides to telomeres [62]. For normal adult cells, telomerase is typically inactive [62]. Activating mutations in the TERT promoter are frequently reported in grade IV astrocytomas (up to 85% of GBM) Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 5 of 21 division prompts telomere truncation until its depletion, which provokes cell dormancy or death [62]. Telomerase reverse transcriptase (TERT) is a subunit of telomerase, an enzyme that inserts additional nucleotides to telomeres [62]. For normal adult cells, telomerase is typically inactive [62]. Activating mutations in the TERT promoter are frequently reported in grade IV astrocytomas (up to 85% of GBM) and grade 2/3 oligodendrogliomas (close to 80%) [62–64]. TERT mutations are strongly Int. J. Mol. Sci. 2018, 19, 1765 5 of 23 correlated with 1p19q co-deletion, but not with either IDH mutations or ATRX loss [64]. Comparisons of groups based on the statuses of IDH, 1p19q, and TERT revealed that TERT mutation bestowsand grade better 2/3 outcomes oligodendrogliomas in gliomas with (close TERT to 80%) mutant/ [62–64IDH]. TERTmutation/1p19q mutations co-d are stronglyeletion, but correlated poorer survivalwith 1p19q in co-deletion,GBM with butTERT not withmutant/ eitherIDHIDH mutationmutations without or ATRX 1p19qloss co-deletion [64]. Comparisons [63]. Currently, of groups detectionbased on theof TERT statuses mutations of IDH, is 1p19q, completed and TERT via methyl-specific revealed that TERT PCR;mutation in addition, bestows rapid better intraoperative outcomes testingin gliomas has been with reportedTERT mutant/ [65]. IDH mutation/1p19q co-deletion, but poorer survival in GBM with TERT mutant/IDH mutation without 1p19q co-deletion [63]. Currently, detection of TERT mutations 8.is Acid completed Ceramidase via methyl-specific (ASAH1) as PCR;a Druggable in addition, Target rapid to Combat intraoperative Multiple testing Therapy-Resistant has been reported [65]. Cancers 8. Acid Ceramidase (ASAH1) as a Druggable Target to Combat Multiple Therapy-Resistant Cancers ASAH1, initially discovered in rat brain homogenates and further characterized and purified from ASAH1human, initiallyurine in discovered 1995, is a in lysosomal rat brain homogenatescysteine amidase and further that catalyzes characterized the transformation and purified from of ceramidehuman urine into insphingosine 1995, is a lysosomal and free fatty cysteine acid (Figure amidase 2) that [66–72]. catalyzes Following the transformation this, sphingosine of ceramide kinase 1 (intoSPHK1 sphingosine) or 2 (SPHK2 and free) phosphorylates fatty acid (Figure sphingosine2)[ 66–72]. Followingto produce this, sphingosine-1-phosphate sphingosine kinase 1 (SPHK1 (S1P),) whichor 2 (SPHK2 promotes) phosphorylates GBM invasiveness sphingosine via the to upregulation produce sphingosine-1-phosphate of the urokinase plasminogen (S1P), which activator, promotes its receptor,GBM invasiveness and the viapro-invasive the upregulation molecule of theCCN1 urokinase (cysteine-rich plasminogen angiogenic activator, protein its receptor, 61) (Figure and the2) [69,72–74].pro-invasive On molecule the otherCCN1 hand,(cysteine-rich high levels of angiogenic ceramides, protein carrying 61) fatty (Figure acid2 )[side69 ,chains72–74]. ranging On the otherfrom 14hand, to 26 high carbons levels and of ceramides,generated via carrying the action fatty of acid ceramide side chains synthases ranging (CerS from), promote 14 to 26 apoptosis carbons and in cellsgenerated that have via the undergone action of ceramideradio- and synthases chemotherapy (CerS), via promote the release apoptosis of cytochrome in cells that c, have leading undergone to the activationradio- and of chemotherapy caspase-9 and viacaspase-3 the release [69–71,75–79]. of cytochrome Since its c, products leading toare the involved activation in the of regulation caspase-9 ofand cell caspase-3 proliferation, [69–71 multiple,75–79]. Sincestudies its have products linked are ASAH1 involved to inmultiple the regulation cancers ofsuch cell as proliferation, melanoma, acutemultiple myeloid studies leukemia have linked (AML),ASAH1 and tocolon multiple and prostate cancers cancers such as melanoma,[80–84]. ASAH1 acute has myeloid been proposed leukemia as(AML), an emerging and colon drug and target prostate in AML cancers [85]. [Interestingly,80–84]. ASAH1 over-expressionhas been proposed of ASAH1 asan in emergingprostate cancer drug promotestarget in AML resistance [85]. Interestingly, to chemothera over-expressionpy. Prostate cancer of ASAH1 upregulatesin prostate ASAH1 cancer following promotes resistanceradiation, whichto chemotherapy. was described Prostate as a mechanism cancer upregulates enablingASAH1 the cancerfollowing to survive radiation, radiation which [86]. was Consequently, described as whena mechanism the activity enabling of ASAH1 the cancer is suppressed to survive radiationwith an ASAH1 [86]. Consequently, inhibitor named when B13, the activitythe cells of becomeASAH1 moreis suppressed sensitive with to chemotherapy an ASAH1 inhibitor and radiation named as B13, a result the cellsof the become accumulation more sensitive of intracellular to chemotherapy ceramide upand to radiation cytotoxic as alevels, result ofinducing the accumulation apoptosis of [81, intracellular87,88]. Similarly, ceramide the up toacid cytotoxic ceramidase levels, inhibitor inducing ceranib-2apoptosis also [81, 87has,88 activity]. Similarly, against the the acid growth ceramidase of the breast inhibitor cancer ceranib-2 cell lines also MCF-7 has activity and MDA against MB-231 the viagrowth the of activation the breast cancerof stress-activated cell lines MCF-7 prot andein MDA kinase/c-Jun MB-231 via theN-terminal activation kinase of stress-activated and p38 mitogen-activatedprotein kinase/c-Jun protein N-terminal kinase kinase apoptotic and p38pathways mitogen-activated and the inhibition protein kinaseof the apoptoticAkt pathway pathways [89]. Theand theWnt inhibition/β-cateninof signaling the Aktpathway pathway [89 appears]. The Wnt to /playβ-catenin a rolesignaling in suppressing pathway the appears proliferation to play a and role metastaticin suppressing potential the proliferation of cervical cancers and metastatic when thes potentiale tumors of cervical were treated cancers with when a recently these tumors identified were ASAH1treated withinhibitor a recently called identified carmofurASAH1 [90,91].inhibitor The clinical called application carmofur [of90 ,carmofur91]. The clinical has been application attempted of andcarmofur it offered has beenbenefits attempted when carmofur and it offered was used benefits an ad whenjuvant carmofur in patients was with used early an adjuvant breast cancer in patients in a postoperativewith early breast setting cancer [92]. in a postoperative setting [92].

Figure 2. AA schematic schematic diagram diagram of of the the sphingolipid sphingolipid signaling signaling pathway, pathway, demonstrating the conversion of ceramides into sphingosine by acid ceramidase ( ASAH1)) and the subsequent transformation of sphingosine into sphingos sphingosine-1-phosphateine-1-phosphate by sphingosine kinase 1 or 2 (SPHK1,, SPHK2).).

9. ASAH1-Induced Radioresistance in GBM The sphingolipid pathway was initially implicated in GBM in several studies, by showing that S1P augments the migratory response of the GBM cell line U87MG and that S1P level is significantly higher in GBM tissues compared to the normal gray matter [93,94]. We provided further evidence of the important role that the sphingolipid pathway plays in GBM. We showed that ASAH1 level was negatively correlated with GBM survival [95]. To study the role ASAH1 plays in radioresistant GBM, we developed a stable radioresistant GBM model, in which U87 GBM cells were irradiated, and the Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 21

9. ASAH1-Induced Radioresistance in GBM The sphingolipid pathway was initially implicated in GBM in several studies, by showing that S1P augments the migratory response of the GBM cell line U87MG and that S1P level is significantly higher in GBM tissues compared to the normal gray matter [93,94]. We provided further evidence of the important role that the sphingolipid pathway plays in GBM. We showed that ASAH1 level was negatively correlated with GBM survival [95]. To study the role ASAH1 plays in radioresistant GBM, weInt. J.developed Mol. Sci. 2018 a, stable19, 1765 radioresistant GBM model, in which U87 GBM cells were irradiated, and6 ofthe 23 surviving cells were perpetuated [96]. In this model, we demonstrated that intracellular ASAH1 was upregulated, and its secretion into extracellular space was also increased in the adult GBM cell line surviving cells were perpetuated [96]. In this model, we demonstrated that intracellular ASAH1 was U87 and in the pediatric GBM cell line SJGBM2, suggesting that ASAH1 confers radioresistance to upregulated, and its secretion into extracellular space was also increased in the adult GBM cell line U87 GBM (Figure 3) [96,97]. Our histochemistry data utilizing patient GBM tissues revealed higher levels and in the pediatric GBM cell line SJGBM2, suggesting that ASAH1 confers radioresistance to GBM of ASAH1 in irradiated tissues compared to control tissues [96]. We suggested that ASAH1 may (Figure3)[ 96,97]. Our histochemistry data utilizing patient GBM tissues revealed higher levels of decrease the overall GBM survival and promote recurrence, which is inevitable, by enhancing the ASAH1 in irradiated tissues compared to control tissues [96]. We suggested that ASAH1 may decrease survival of irradiated GBMs via the upregulation of ASAH1, leading to decreasing levels of the overall GBM survival and promote recurrence, which is inevitable, by enhancing the survival of proapoptotic ceramide molecules and increasing levels of prosurvival S1P molecules (Figure 3) [96]. irradiated GBMs via the upregulation of ASAH1, leading to decreasing levels of proapoptotic ceramide Despite being resistant to radiation, these cells remained sensitive to the ASAH1 inhibitor carmofur, molecules and increasing levels of prosurvival S1P molecules (Figure3)[ 96]. Despite being resistant to albeit displaying a slightly higher IC50 value [96]. More importantly, ASAH1 inhibitors have been radiation, these cells remained sensitive to the ASAH1 inhibitor carmofur, albeit displaying a slightly proposed as radiosensitizers, on the basis of studies that illustrated a greater suppression of the higher IC value [96]. More importantly, ASAH1 inhibitors have been proposed as radiosensitizers, growth of50 U87 and prostate cancer xenografts when treated with both conventional radiation on the basis of studies that illustrated a greater suppression of the growth of U87 and prostate cancer therapy and ASAH1 inhibitors [87,98]. Carmofur is the only ASAH1 inhibitor that has been used xenografts when treated with both conventional radiation therapy and ASAH1 inhibitors [87,98]. clinically to treat colorectal cancers [99–101]. However, carmofur has several issues that need to be Carmofur is the only ASAH1 inhibitor that has been used clinically to treat colorectal cancers [99–101]. addressed before it can be more widely used. It has very low solubility in aqueous solution, an However, carmofur has several issues that need to be addressed before it can be more widely used. intravenous formula is unavailable, and the extent to which it can penetrate the blood–brain barrier It has very low solubility in aqueous solution, an intravenous formula is unavailable, and the extent to is poorly understood [91]. One strategy to improve the solubility of carmofur is to take advantage of which it can penetrate the blood–brain barrier is poorly understood [91]. One strategy to improve the the recently solved crystal structure of acid ceramidase to help predict how carmofur would fit in its solubility of carmofur is to take advantage of the recently solved crystal structure of acid ceramidase to active site and perform appropriate modifications to allow the drug to be both more soluble and help predict how carmofur would fit in its active site and perform appropriate modifications to allow potent [102]. Another strategy to combat radioresistance induced by secretion of ASAH1 is to induce the drug to be both more soluble and potent [102]. Another strategy to combat radioresistance induced the immune system to produce autoantibodies against extracellular ASAH1. The benefit of by secretion of ASAH1 is to induce the immune system to produce autoantibodies against extracellular developing auto-ASAH1 antibodies was demonstrated in melanoma patients. The auto anti-ASAH1 ASAH1. The benefit of developing auto-ASAH1 antibodies was demonstrated in melanoma patients. antibodies protected the melanoma patients from lymph node metastasis, and the loss of these The auto anti-ASAH1 antibodies protected the melanoma patients from lymph node metastasis, antibodies could result in melanoma progression [103]. A strategy to promote the development of and the loss of these antibodies could result in melanoma progression [103]. A strategy to promote auto-ASAH1 antibodies is by immunizing patients against ASAH1, and this may mitigate the the development of auto-ASAH1 antibodies is by immunizing patients against ASAH1, and this may proliferation and invasion of radioresistant GBM. Further study is needed to examine whether the mitigate the proliferation and invasion of radioresistant GBM. Further study is needed to examine auto-ASAH1 antibodies can cross the blood–brain barrier, as there is a paucity of data available whether the auto-ASAH1 antibodies can cross the blood–brain barrier, as there is a paucity of data regarding the benefit of auto-antibodies in treating neurological diseases. available regarding the benefit of auto-antibodies in treating neurological diseases.

Figure 3. Schematic diagram is shown describing the molecular changes occurring in a glioblastoma (GBM) cell following radiation: increased secretion of ASAH1, increased intracellular levels of ASAH1 and S1P and decreased level of ceramides. ↑: Upregulation, ↓: Downregulation

10. Identification of Novel Drug Targets to Combat Radioresistant GBM The current standard treatment regimen for GBM includes maximal safe surgical resection, followed by radiation therapy combined with concomitant and adjuvant temozolomide [30,104]. Int. J. Mol. Sci. 2018, 19, 1765 7 of 23

However, recurrence of GBM—characterized by radioresistance—remains inevitable [105,106]. The absence of a major progress in the treatment of GBM maybe a result of our poor understanding of both GBM tumor biology and the mechanisms underlying the acquirement of treatment resistance in recurrent GBMs. In support of this view, very little data about the radiation effects on global gene expression at the messenger ribonucleic acid (mRNA) level in a stable radioresistant GBM model are available. Ma et al., in their transcriptome analysis of glioma within hours following irradiation, suggested that the development of radioresistance of glioma may be due to the inactivation of early proapoptotic molecules and to the late activation of antiapoptotic genes [107]. To identify radiation-responsive genes that may enable GBM cells to acquire resistance to radiation, we performed complete RNA sequencing (RNA-seq) of control tissues and our recently established stable, radioresistant U87-based GBM model [96,108]. Our study revealed that the aberrant gene expression observed in irradiated U87-10gy cells regarded, in particular, genes involved in enhancing tumor malignancy and invasion. In irradiated U87-10gy cells, we observed the upregulation of antiapoptotic genes (BNIP3 and SOD2), of genes promoting epithelial to mesenchymal transition, of genes with metalloendopeptidase activity, and of genes involved in the response to hypoxia (Tables1 and2)[ 108]. Metalloproteases are known to promote tumor invasion and metastasis of many cancers by degrading the extracellular matrix [109,110]. MME, MMP2, MMP3, MMP7, MMP12, ADAM9, and ADAM12 were shown to be upregulated in radioresistant GBMs (Tables1 and2)[ 108]. Epithelial to mesenchymal transition, a process characterized by increased cell motility and resistance to chemo- and radiotherapy, is typically induced by TGFB3, which was also upregulated in irradiated U87-10gy cells [108,111,112]. Hypoxia, which is frequent in GBM, induces hypoxia-inducible factor 1-alpha (HIF-1α) and carbonic anhydrase 9 expressions, which in turn promote angiogenesis, migration, cell survival, proliferation, epithelial to mesenchymal transition, and radio- and chemoresistance [111,113,114]. HIF-1α and carbonic anhydrase 9 were upregulated in irradiated GBM cells [108]. On the other hand, we found that the downregulated genes were enriched in tumor suppressors, in genes positively regulating the immune response, in genes involved in p53-dependent apoptosis, and in cell adhesion genes. Suppressing the apoptotic potential through gene expression regulation in the irradiated cells was a proposed mechanism that explained the radioresistant nature of the irradiated GBM cells [107,108]. Many apoptotic genes discovered in our study were known to play major roles in attenuating GBM apoptosis, especially, BBC3, DCC, BEX2, CASP1, IL1B, and SFRP2 [115–120]. GBM cells produce an immunosuppressive microenvironment to escape immune surveillance and enhance their own survival, and this can be accomplished through the secretion of transforming growth factor β (TGF-β) to block T cell activation and proliferation [121]. We identified many other downregulated genes involved in the activation of the immune system, especially genes mediating T cell antigen processing and presentation that may enable immune evasion in the radioresistant GBM cells [108]. Considering these global alterations of multiple biological pathways observed in irradiated GBM cells, an effective treatment targeting radioresistant GBM may require a cocktail containing multiple agents targeting multiple implicated pathways in order to have a chance to make a substantial impact on improving the overall GBM survival. Int. J. Mol. Sci. 2018, 19, 1765 8 of 23

Table 1. Upregulated genes of selected enriched gene ontology categories following irradiation are shown on the basis of sets of statistically significant changes (p < 0.05) [108].

GO:0006954: Inflammatory Response GO:0000187: Activation of MAPK Activity CCL26 C–C motif chemokine ligand 26 (CCL26) anaplastic lymphoma receptor tyrosine kinase (ALK) CCL3 C–C motif chemokine ligand 3 (CCL3) chondroitin sulfate proteoglycan 4 (CSPG4) CXCL8 C–X–C motif chemokine ligand 8 (CXCL8) dual specificity phosphatase 7 (DUSP7) GPR68 G protein-coupled receptor 68 (GPR68) formyl peptide receptor 1 (FPR1) NFKBID NFKB inhibitor delta (NFKBID) transforming growth factor beta 3 (TGFB3) TNFAIP3 TNF alpha-induced protein 3 (TNFAIP3) tumor protein p73 (TP73) TNFRSF10D TNF receptor superfamily member 10d (TNFRSF10D) TNIP3 TNFAIP3 interacting protein 3 (TNIP3) GO:0004222: Metalloendopeptidase Activity XCR1 X–C motif chemokine receptor 1 (XCR1) ADAM metallopeptidase domain 12 (ADAM12) BDKRB1 bradykinin receptor B1 (BDKRB1) ADAM metallopeptidase domain 19 (ADAM19) BDKRB2 bradykinin receptor B2 (BDKRB2) ADAM metallopeptidase with thrombospondin type 1 motif 1 (ADAMTS1) CHST4 carbohydrate sulfotransferase 4 (CHST4) ADAM metallopeptidase with thrombospondin type 1 motif 14 (ADAMTS14) C3 complement C3 (C3) bone morphogenetic protein 1 (BMP1) FPR1 formyl peptide receptor 1 (FPR1) matrix metallopeptidase 12 (MMP12) GBP5 guanylate binding protein 5 (GBP5) matrix metallopeptidase 3 (MMP3) IL24 interleukin 24 (IL24) matrix metallopeptidase 7 (MMP7) IL36B interleukin 36, beta (IL36B) membrane metalloendopeptidase (MME) NFATC4 nuclear factor of activated T-cells 4 (NFATC4) teashirt zinc finger homeobox 2 (TSHZ2) PTGER2 prostaglandin E receptor 2 (PTGER2) SDC1 syndecan 1 (SDC1) GO:0071356: Cellular Response to Tumor Necrosis Factor ZC3H12A zinc finger CCCH-type-containing 12A (ZC3H12A) C–C motif chemokine ligand 26 (CCL26) C–C motif chemokine ligand 3 (CCL3) GO:0010718: Positive Regulation of Epithelial to Mesenchymal Transition C–X–C motif chemokine ligand 8 (CXCL8) BAMBI BMP and activin membrane-bound inhibitor (BAMBI) ankyrin repeat domain 1 (ANKRD1) GLIPR2 GLI pathogenesis related 2 (GLIPR2) collagen type I alpha 1 chain (COL1A1) Int. J. Mol. Sci. 2018, 19, 1765 9 of 23

Table 1. Cont.

AXIN2 axin 2 (AXIN2) endothelin 1 (EDN1) COL1A1 collagen type I alpha 1 chain (COL1A1) hyaluronan synthase 2 (HAS2) TGFB3 transforming growth factor beta 3 (TGFB3) periostin (POSTN) GO:0016477: Cell Migration BAMBI BMP and activin membrane-bound inhibitor (BAMBI) GO:0044344: Cellular Response to Fibroblast Growth Factor EPHA3 EPH receptor A3 (EPHA3) C–X–C motif chemokine ligand 8 (CXCL8) EPHB3 EPH receptor B3 (EPHB3) collagen type I alpha 1 chain (COL1A1) ERG ERG, ETS transcription factor (ERG) periostin (POSTN) WWC1 WW and C2 domain containing 1 (WWC1) snail family transcriptional repressor 2 (SNAI2) BDKRB1 bradykinin receptor B1 (BDKRB1) CSPG4 chondroitin sulfate proteoglycan 4 (CSPG4) GO:0071560: Cellular Response to Transforming Growth Factor Beta COL5A1 collagen type V alpha 1 chain (COL5A1) ankyrin repeat domain 1 (ANKRD1) FSCN1 fascin actin-bundling protein 1 (FSCN1) collagen type I alpha 1 chain (COL1A1) LCP1 lymphocyte cytosolic protein 1 (LCP1) endothelin 1 (EDN1) PODXL podocalyxin like (PODXL) periostin (POSTN) PSG2 pregnancy specific beta-1-glycoprotein 2 (PSG2) phosphodiesterase 3A (PDE3A) SDC1 syndecan 1 (SDC1) GO:0001525: Angiogenesis ribonuclease A family member 1, pancreatic (RNASE1) CXCL8 C–X–C motif chemokine ligand 8 (CXCL8) ribonuclease A family member 2 (RNASE2) EPHB3 EPH receptor B3 (EPHB3) EPHB4 EPH receptor B4 (EPHB4) GO:0090263: Positive Regulation of Canonical Wnt Signaling Pathway ACKR3 atypical chemokine receptor 3 (ACKR3) BMP and activin membrane bound inhibitor (BAMBI) CSPG4 chondroitin sulfate proteoglycan 4 (CSPG4) R-spondin 3 (RSPO3) COL8A1 collagen type VIII alpha 1 chain (COL8A1) SRY-box 4 (SOX4) NRXN3 neurexin 3 (NRXN3) axin 2 (AXIN2) NDNF neuron-derived neurotrophic factor (NDNF) collagen type I alpha 1 chain (COL1A1) NRP2 neuropilin 2 (NRP2) distal-less homeobox 5 (DLX5) Int. J. Mol. Sci. 2018, 19, 1765 10 of 23

Table 1. Cont.

GO:0008283: Cell Proliferation 4-aminobutyrate aminotransferase (ABAT) SERPINE1 serpin family E member 1 (SERPINE1) leucine rich repeat containing G protein-coupled receptor 4 (LGR4) ZC3H12A zinc finger CCCH-type containing 12A (ZC3H12A) E2F8 E2F transcription factor 8 (E2F8) BCL2 interacting protein 3 (BNIP3) ERG ERG, ETS transcription factor (ERG) carbonic anhydrase 9 (CA9) ROS1 ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) cytochrome P450 family 1 subfamily A member 1 (CYP1A1) ALK anaplastic lymphoma receptor tyrosine kinase (ALK) egl-9 family hypoxia inducible factor 3 (EGLN3) AXIN2 axin 2 (AXIN2) lysyl oxidase like 2 (LOXL2) CDC25A cell division cycle 25A (CDC25A) mucin 1, cell surface associated (MUC1) CSPG4 chondroitin sulfate proteoglycan 4 (CSPG4) periostin (POSTN) CYP1A1 cytochrome P450 family 1 subfamily A member 1 (CYP1A1) transforming growth factor beta 3 (TGFB3) DLX5 distal-less homeobox 5 (DLX5) FSCN1 fascin actin-bundling protein 1 (FSCN1) FGF5 fibroblast growth factor 5 (FGF5) GRPR gastrin releasing peptide receptor (GRPR) MYH10 myosin heavy chain 10 (MYH10) PDK1 pyruvate dehydrogenase kinase 1 (PDK1) UHRF1 ubiquitin like with PHD and ring finger domains 1 (UHRF1) GO:0016049: Cell Growth ROS1 ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) EDN1 endothelin 1 (EDN1) IL7R interleukin 7 receptor (IL7R) NDNF neuron-derived neurotrophic factor (NDNF) TGFB3 transforming growth factor beta 3 (TGFB3) Int. J. Mol. Sci. 2018, 19, 1765 11 of 23

Table 2. Downregulated genes of selected enriched gene ontology categories following irradiation are shown on the basis of sets of statistically significant changes (p < 0.05) [108].

GO:0006915: Apoptotic Process GO:0008152: Metabolic Process BCL2 binding component 3 (BBC3) 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) DCC netrin 1 receptor (DCC) UDP glucuronosyltransferase family 1 member A1 (UGT1A1) PYD and CARD domain-containing (PYCARD) UDP glucuronosyltransferase family 1 member A10 (UGT1A10) TNF receptor-associated factor 5 (TRAF5) UDP glucuronosyltransferase family 1 member A3 (UGT1A3) XIAP-associated factor 1 (XAF1) UDP glucuronosyltransferase family 1 member A4 (UGT1A4) Brain-expressed X-linked 2 (BEX2) UDP glucuronosyltransferase family 1 member A5 (UGT1A5) caspase 1 (CASP1) UDP glucuronosyltransferase family 1 member A6 (UGT1A6) cathepsin H (CTSH) UDP glucuronosyltransferase family 1 member A7 (UGT1A7) complement C5a receptor 1 (C5AR1) UDP glucuronosyltransferase family 1 member A8 (UGT1A8) engulfment and cell motility 1 (ELMO1) UDP glucuronosyltransferase family 1 member A9 (UGT1A9) interleukin 1 beta (IL1B) acyl-CoA synthetase medium-chain family member 5 (ACSM5) mitogen-activated protein kinase kinase 6 (MAP2K6) acyl-CoA synthetase short-chain family member 1 (ACSS1) nuclear receptor subfamily 4 group A member 1 (NR4A1) acyl-CoA synthetase short-chain family member 3 (ACSS3) phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) glutathione S-transferase mu 5 (GSTM5) secreted frizzled related protein 2 (SFRP2) haloacid dehalogenase-like hydrolase domain-containing 3 (HDHD3) tyrosyl-tRNA synthetase (YARS) lipase E, hormone sensitive type (LIPE) mannosidase alpha class 1C member 1 (MAN1C1) GO:0072332: Intrinsic Apoptotic Signaling Pathway by p53 Class Mediator PERP, TP53 apoptosis effector (PERP) GO:0007155: Cell Adhesion PYD and CARD domain-containing (PYCARD) CD22 molecule (CD22) phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) CD9 molecule (CD9) zinc finger matrin-type 1 (ZMAT1) EPH receptor A4 (EPHA4) zinc finger protein 385D (ZNF385D) adhesion G protein-coupled receptor G1 (ADGRG1) amelotin (AMTN) GO:2000406: Positive Regulation of T Cell Migration basal cell adhesion molecule (Lutheran blood group) (BCAM) PYD and CARD domain-containing (PYCARD) brevican (BCAN) Int. J. Mol. Sci. 2018, 19, 1765 12 of 23

Table 2. Cont.

TNF receptor superfamily member 14 (TNFRSF14) cadherin 11 (CDH11) integrin subunit alpha 4 (ITGA4) collagen type IV alpha 6 chain (COL4A6) fasciculation and elongation protein zeta 1 (FEZ1) GO:0002457: T Cell Antigen Processing and Presentation fibulin 7 (FBLN7) intercellular adhesion molecule 1 (ICAM1) hemicentin 2 (HMCN2) raftlin, lipid raft linker 1 (RFTN1) hyaluronan synthase 1 (HAS1) integrin subunit alpha 11 (ITGA11) GO:0002282: Microglial Cell Activation Involved in Immune Response integrin subunit alpha 2 (ITGA2) interleukin 33 (IL33) integrin subunit alpha 4 (ITGA4) toll like receptor 3 (TLR3) integrin subunit alpha L (ITGAL) integrin subunit beta 8 (ITGB8) GO:0007165: Signal Transduction intercellular adhesion molecule 1 (ICAM1) ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2 (ARAP2) junction plakoglobin (JUP) G kinase anchoring protein 1 (GKAP1) laminin subunit alpha 2 (LAMA2) G protein subunit gamma 11 (GNG11) laminin subunit alpha 3 (LAMA3) GULP, engulfment adaptor PTB domain-containing 1 (GULP1) ninjurin 1 (NINJ1) KIT proto-oncogene receptor tyrosine kinase (KIT) protein kinase C epsilon (PRKCE) MX dynamin like GTPase 1 (MX1) protein kinase, X-linked (PRKX) NDP, norrin cystine knot growth factor (NDP) protocadherin 17 (PCDH17) NLR family pyrin domain-containing 12 (NLRP12) sphingosine-1-phosphate receptor 1 (S1PR1) NLR family pyrin domain-containing 3 (NLRP3) trophoblast glycoprotein (TPBG) PYD and CARD domain-containing (PYCARD) versican (VCAN) Ras association domain family member 9 (RASSF9) Rho family GTPase 2 (RND2) GO:0045746: Negative Regulation Of Notch Signaling Pathway SPARC-related modular calcium binding 1 (SMOC1) ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1) TNF receptor-associated factor 5 (TRAF5) MAGE family member A1 (MAGEA1) TNF receptor superfamily member 11b (TNFRSF11B) Hes-related family bHLH transcription factor with YRPW motif 1 (HEY1) Int. J. Mol. Sci. 2018, 19, 1765 13 of 23

Table 2. Cont.

amyloid beta precursor protein binding family B member 1 interacting protein (APBB1IP) maternally expressed 3 (non-protein coding) (MEG3) androgen receptor (AR) basal cell adhesion molecule (Lutheran blood group) (BCAM) GO:0010759: Positive Regulation of Macrophage Chemotaxis calcitonin-related polypeptide beta (CALCB) chemerin chemokine-like receptor 1 (CMKLR1) caspase 1 (CASP1) complement C5a receptor 1 (C5AR1) chimerin 1 (CHN1) tumor necrosis factor superfamily member 18 (TNFSF18) complement C5a receptor 1 (C5AR1) fibroblast growth factor 18 (FGF18) GO:0006351: Transcription, DNA-Templated fibroblast growth factor 7 (FGF7) CREB3 regulatory factor (CREBRF) growth differentiation factor 15 (GDF15) DNA damage inducible transcript 3 (DDIT3) inositol-trisphosphate 3-kinase A (ITPKA) E2F transcription factor 7 (E2F7) insulin like growth factor binding protein 1 (IGFBP1) HKR1, GLI-Kruppel zinc finger family member (HKR1) insulin like growth factor binding protein 5 (IGFBP5) Kruppel-like factor 2 (KLF2) integrin subunit alpha L (ITGAL) Kruppel-like factor 9 (KLF9) interleukin 1 beta (IL1B) MAGE family member A1 (MAGEA1) interleukin 15 receptor subunit alpha (IL15RA) MAX dimerization protein 1 (MXD1) junction plakoglobin (JUP) MLX interacting protein like (MLXIPL) mitogen-activated protein kinase 10 (MAPK10) NLR family pyrin domain-containing 3 (NLRP3) mitogen-activated protein kinase kinase 6 (MAP2K6) RAR related orphan receptor B (RORB) nuclear receptor subfamily 2 group F member 1 (NR2F1) SATB homeobox 1 (SATB1) nuclear receptor subfamily 4 group A member 1 (NR4A1) T-box 3 (TBX3) nuclear receptor subfamily 4 group A member 2 (NR4A2) TGFB-induced factor homeobox 2 like, X-linked (TGIF2LX) phosphodiesterase 10A (PDE10A) ZFP14 zinc finger protein (ZFP14) phosphodiesterase 1A (PDE1A) androgen receptor (AR) phosphodiesterase 4D (PDE4D) endoplasmic reticulum to nucleus signaling 1 (ERN1) placental growth factor (PGF) forkhead box P2 (FOXP2) Int. J. Mol. Sci. 2018, 19, 1765 14 of 23

Table 2. Cont.

plasminogen activator, urokinase (PLAU) hair growth associated (HR) protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) hes-related family bHLH transcription factor with YRPW motif 1 (HEY1) protein kinase C epsilon (PRKCE) homeobox B7 (HOXB7) protein kinase C zeta (PRKCZ) homeobox B8 (HOXB8) ras-related dexamethasone induced 1 (RASD1) homeobox B9 (HOXB9) ribosomal protein S6 kinase A2 (RPS6KA2) interleukin 33 (IL33) ribosomal protein S6 kinase A6 (RPS6KA6) iroquois homeobox 5 (IRX5) secreted and transmembrane 1 (SECTM1) leucine zipper tumor suppressor 1 (LZTS1) single Ig and TIR domain-containing (SIGIRR) mitogen-activated protein kinase kinase 6 (MAP2K6) thrombomodulin (THBD) myelin expression factor 2 (MYEF2) toll like receptor 3 (TLR3) neuronal PAS domain protein 2 (NPAS2) transducin-like enhancer of split 1 (TLE1) nuclear factor I B (NFIB) tumor necrosis factor superfamily member 10 (TNFSF10) nuclear protein 1, transcriptional regulator (NUPR1) tumor necrosis factor superfamily member 13b (TNFSF13B) nuclear receptor coactivator 7 (NCOA7) tumor necrosis factor superfamily member 18 (TNFSF18) nuclear receptor subfamily 2 group F member 1 (NR2F1) tyrosyl-tRNA synthetase (YARS) nuclear receptor subfamily 4 group A member 1 (NR4A1) unc-5 netrin receptor B (UNC5B) nuclear receptor subfamily 4 group A member 2 (NR4A2) unc-5 netrin receptor C (UNC5C) protein kinase AMP-activated catalytic subunit alpha 2 (PRKAA2) very low density lipoprotein receptor (VLDLR) thyroid hormone receptor beta (THRB) transducin-like enhancer of split 1 (TLE1) tribbles pseudokinase 3 (TRIB3) tumor protein p63 (TP63) twist family bHLH transcription factor 2 (TWIST2) vestigial-like family member 2 (VGLL2) visual system homeobox 1 (VSX1) zinc finger and SCAN domain containing 16 (ZSCAN16) zinc finger family member 788 (ZNF788) Int. J. Mol. Sci. 2018, 19, 1765 15 of 23

Table 2. Cont.

zinc finger protein 117 (ZNF117) zinc finger protein 138 (ZNF138) zinc finger protein 20 (ZNF20) zinc finger protein 273 (ZNF273) zinc finger protein 28 (ZNF28) zinc finger protein 30 (ZNF30) zinc finger protein 320 (ZNF320) zinc finger protein 354B (ZNF354B) zinc finger protein 396 (ZNF396) zinc finger protein 415 (ZNF415) zinc finger protein 419 (ZNF419) zinc finger protein 433 (ZNF433) zinc finger protein 44 (ZNF44) zinc finger protein 442 (ZNF442) zinc finger protein 443 (ZNF443) zinc finger protein 468 (ZNF468) zinc finger protein 521 (ZNF521) zinc finger protein 525 (ZNF525) zinc finger protein 528 (ZNF528) zinc finger protein 549 (ZNF549) zinc finger protein 563 (ZNF563) zinc finger protein 572 (ZNF572) zinc finger protein 577 (ZNF577) zinc finger protein 625 (ZNF625) zinc finger protein 649 (ZNF649) zinc finger protein 674 (ZNF674) zinc finger protein 680 (ZNF680) zinc finger protein 71 (ZNF71) Int. J. Mol. Sci. 2018, 19, 1765 16 of 23

Table 2. Cont.

zinc finger protein 761 (ZNF761) zinc finger protein 765 (ZNF765) zinc finger protein 792 (ZNF792) zinc finger protein 799 (ZNF799) zinc finger protein 806 (ZNF806) zinc finger protein 816 (ZNF816) zinc finger protein 83 (ZNF83) zinc finger protein 845 (ZNF845) zinc finger protein 85 (ZNF85) zinc finger protein 883 (ZNF883) zinc finger protein 888 (ZNF888) zinc finger with KRAB and SCAN domains 7 (ZKSCAN7) Int. J. Mol. Sci. 2018, 19, 1765 17 of 23

Acknowledgments: This study was funded by the Musella Foundation Grant and the Department of Neurosurgery Larson Endowment Grant. Conflicts of Interest: The authors declare no conflicts of interest.

Abbreviations

5-ALA 5-aminolevulinic acid ASAH1 acid ceramidase AML acute myeloid leukemia CerS ceramide synthases DMG N-dimethyl glycine GBM glioblastoma IDH isocitrate dehydrogenase MGMT O6-methylguanine-methyltransferase MRI magnetic resonance imaging S1P sphingosine-1-phosphate SPHK1 sphingosine kinase 1 SPHK2 sphingosine kinase 2 TTF tumor-treating field

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